In vitro fertilization, embryo culture and embryo transplantation and the like are important techniques in a wide range of fields such as the improvement and production of animals with a high economic value, regenerative medicine and reproductive medicine. In in vitro fertilization and embryo culture, gametes or embryos are treated in vitro. Until now, various media for embryo culture have been developed, such as sequential culture media which are made to imitate changes in the environment of peripheral body fluids during embryonic development, serum media which are supplemented with serum and serum-free simple media called “single medium” which can be easily treated. When using a single medium, medium exchange in accordance with the developmental stage of embryos as a sequential culture media is not required. When using a serum-free medium, risks which are caused in serum media can be reduced, such as quality variation, and virus contamination because of products derived from organisms.
As one of serum-free single media, KSOM medium is known (Proc Natl Acad Sci USA 2007; 104(36): 14289-14293 and Manipulating the Mouse Embryo, THIRD EDITION 2005; 149-193.). In embryos cultured in the KSOM medium, the number of cells in blastocysts is increased as compared to that in embryos cultured in a medium before the appearance of KSOM (Fertil Steril 2006; 86: 1252-1265.), however, the number does not reach to the number of cells in blastocysts developed in vivo (Biol Reprod 1994; 50: 1027-1033 and Reproduction 2009; 137: 271-283.).
It is known that amino acids act as biosynthetic precursors, energy sources, organic osmolytes, intracellular pH buffer substances, antioxidants, chelators and the like, and the addition of amino acids to media for embryo culture effectively acts on embryonic development (Semin Reprod Med 2000; 18(2): 205-218). The KSOM medium was a medium which contained only glutamine as an amino acid. It has been revealed, however, that a blastocyst development rate, a hatching rate and the number of cells in blastocysts are improved by adding 20 types of amino acids to the KSOM medium (Mol Reprod Dev 1995; 41(2): 232-238).
When developing a medium for culturing human pre-implantation embryos, researches using mouse embryos are recommended (Human Reproduction 1998; 13(4): 173-183 and Human Reproduction Update 2003; 9(6): 557-582). The KSOM medium or KSOMaa medium to which 20 types of amino acids are added has been researched using mouse embryos (Biol Reprod 1994; 50: 1027-1033 and Mol Reprod Dev 1995; 41(2): 232-238). It has been verified, however, that the media are suitable for culturing not only human embryos but also various animal embryos such as bovine, rabbit, rhesus monkey, swine and rat, and the media are now widely used.